rhamnosus GR-1 showed a three- to fourfold induction of NF-κB compared to cells that had Tucidinostat no lactobacilli added. Figure 4 NF-κB augmentation by two different L. rhamnosus strains. Bladder cells were co-stimulated with heat-killed E. coli and viable L. rhamnosus GR-1 or L. rhamnosus GG for 24 h (n = 4). An asterisk denotes significant difference between the two groups (p-values < 0.05). L. rhamnosus GR-1

modified TLR4 expression on bladder cells TLR4 is a crucial protein in the detection of E. coli by epithelial cells, therefore we proceeded by analyzing the levels of TLR4 in bladder cells treated with heat-killed E. coli and L. rhamnosus GR-1. Co-stimulated bladder cells showed increased expression of TLR4 mRNA compared to cells stimulated with E. coli or lactobacilli alone (Figure 5A). Furthermore, immunoblotting using native proteins showed high band intensity in co-stimulated cells suggesting higher TLR4 protein content compared to all other groups (Figure 5B). The effect on TLR4 protein levels was further characterized using confocal laser microscopy.

Control cells and cells stimulated with only E. coli Selonsertib chemical structure or lactobacilli showed no or low TLR expression, whereas cells co-stimulated with both E. coli and L. rhamnosus GR-1 demonstrated a substantial increase in the amount of TLR4 protein (Figure 5C). Figure 5 TLR4 expression in bladder cells after Lactobacillus stimulation. (A) TLR4 qPCR from cells co-stimulated for 3 h with E. coli and L. rhamnosus GR-1 (n = 3). (B) A native immunoblot of TLR4 protein after 24 h stimulation. (C) Confocal microscopy of TLR4 protein (green pixels) after stimulation of T24 cells.

The cells were also stained with DAPI (DNA stain) and Alexa555 phalloidin (actin stain) and pseudo-colored blue and red, respectively. Immunoblot and confocal images Mephenoxalone are representative data from two or more separate experiments. Bars labeled with an asterisk are significantly different from control cells (p-values < 0.05). Polymyxin B suppressed NF-κB augmentation We continued to characterize the role of TLR4 in NF-κB activation by co-stimulation with heat-killed E. coli and lactobacilli. The TLR4 activation in bladder cells was inhibited by pretreatment with polymyxin B, a known inhibitor of LPS-induced TLR4 activation, and thereafter stimulated by E. coli and L. rhamnosus GR-1 (Figure 6). Polymyxin B significantly inhibited NF-κB activation in cells challenged with both E. coli and lactobacilli although it had no significant effect on NF-κB activation in resting cells and on lactobacilli treated cells. The increased NF-κB activation observed during co-stimulation was completely lost after polymyxin B treatment, demonstrating the involvement of LPS and TLR4. Figure 6 NF-κB potentiation is TLR4 dependant. Polymyxin B (PMB) was added to cell culture before stimulation to inhibit TLR4 activation. Cells were stimulated with heat-killed E. coli and viable L. rhamnosus GR-1 for 24 h (n = 3).